There is convincing clinical evidence that the epidermal growth factor receptor (EGFR)-specific monoclonal antibody (mAb), cetuximab, is effective therapy for advanced head and neck squamous cell carcinoma (SCCHN). However, not all patients respond to cetuximab, and clinical responses are not correlated with level of EGFR expression on tumor cells. Thus, there is a need to understand why clinical responses vary among patients, in order to select those most likely to respond to cetuximab therapy. In contrast to EGFR tyrosine kinase inhibitors, the antitumor activity of cetuximab may benefit from its interactions with host's immune system, since cetuximab induces significant killing of SCCHN cells in vitro only in combination with natural killer (NK) cells and monocytes. Furthermore, in an animal model system the antitumor activity of cetuximab is enhanced by the addition of NK cells. This information has provided the rationale for our hypothesis that the antitumor activity of cetuximab is influenced by its ability to trigger an EGFR-specific cellular immune response and by the escape mechanisms SCCHN cells utilize to avoid immune recognition and destruction. In this proposal, we will first characterize in vitro the variables, which are involved in the generation of EGFR-specific cytotoxic T lymphocytes (CTL) by cetuximab. They include the polymorphism of the Fcy receptors expressed by NK cells, the EGFR expression level on SCCHN cells, and the ability of dendritic cells (DC) to cross-present tumor antigens to T cells following loading with SCCHN cells coated with cetuximab. In addition, we will characterize how the immunosuppressive activity of regulatory T cells interferes with the generation of EGFR-specific CTL as well as with their lytic activity and that of NK cells. To assess the clinical relevance of the in vitro results, we will determine how the balance between the induction of an EGFR-specific CTL response triggered by cetuximab and the immune escape mechanisms utilized by SCCHN cells impacts the clinical response to (antitumor activity of) cetuximab in SCCHN patients who will be enrolled in a trial associated with this project. The results derived from this study will have an impact on the clinical application of cetuximab-based immunotherapy in SCCHN patients by identifying predictive immune biomarkers of biological and clinical responses, optimizing the selection of patients to be treated with cetuximabbased immunotherapy and contributing to the design of interventions to enhance its efficacy. Furthermore, the principles defined with cetuximab may be applicable to other TA-specific mAb; therefore, the information derived from this translational proposal may have a broad significance for the clinical application of antibody-based immunotherapy to the treatment of malignant diseases.